Pyridyl analogs of 1-(2-cyano-3,12-dioxooleana-1,9(11)dien-28-oyl) imidazole

ABSTRACT

Pyridyl analogs of 1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl) imidazole and pharmaceutical compositions containing the same are provided.

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/505,327, filed Feb. 21, 2017, which is a 371 application ofPCT/US2015/046845 filed Aug. 26, 2015, which claims benefit of priorityof Provisional Application No. 62/041,802 filed Aug. 26, 2014, thecontents of which are incorporated herein by reference in theirentirety.

BACKGROUND

One of the major needs in cancer prevention is the development ofeffective and safe new agents for chemoprevention. In particular, thereis a need for chemopreventative agents targeted at mechanisms known tobe involved in the process of carcinogenesis. In recent years, there hasbeen a resurgence of interest in the study of mechanisms of inflammationthat relate to carcinogenesis and in the use of such mechanisms as thebasis for development of new chemopreventative agents.

The concept that inflammation and carcinogenesis are related phenomenahas been the subject of many studies that have attempted to link thesetwo processes in a mechanistic fashion (Sporn & Roberts (1986) J. Clin.Invest. 78:329-332; Ohshima & Bartsch (1994) Mutat. Res. 305:253-264).The enzymes that mediate the constitutive synthesis of nitric oxide andprostaglandins from arginine and arachidonate, respectively, haverelative little significance for either inflammation or carcinogenesis.In contrast, inducible nitric oxide synthase (iNOS) and induciblecycloxygenase (COX-2) both have critical roles in the response oftissues to injury or infectious agents (Moncada, et al. (1991)Pharmacol. Rev. 43:109-142; Nathan & Xie (1994) Cell 78:915-918; Siebert& Masferrer (1994) Receptor 4(1):17-23; Tamir & Tannebaum (1996)Biochim. Biophys. Acta 1288:F31-F36). These inducible enzymes areessential components of the inflammatory process, the ultimate repair ofinjury, and carcinogenesis. While physiological activity of iNOS andCOX-2 may provide a definite benefit to the organism, aberrant orexcessive expression of either iNOS or COX-2 has been implicated in thepathogenesis of many disease processes, particularly in chronicdegeneration of the central nervous system, carcinogenesis, septicshock, cardiomyopathy, and rheumatoid arthritis.

Triterpenoids, biosynthesized in plants by the cyclization of squalene,are used for medicinal purposes in many Asian countries; and some, likeursolic and oleanolic acids, are known to be anti-inflammatory andanti-carcinogenic (Huang, et al. (1994) Cancer Res. 54:701-708; Nishino,et al. (1988) Cancer Res. 48:5210-5215). However, the biologicalactivity of these naturally occurring molecules is relatively weak, andtherefore the synthesis of new analogs to enhance their potency has beenundertaken (see, e.g., Honda, et al. (1997) Bioorg. Med. Chem. Lett.7:1623-1628; Honda, et al. (1998) Bioorg Med Chem Lett.8(19):2711-2714).

In this respect, U.S. Pat. No. 6,326,507, U.S. Pat. No. 6,552,075, U.S.Pat. No. 7,288,568, U.S. Pat. No. 7,863,327, U.S. Pat. No. 8,034,955, US2009/0060873, US 2009/0048204, WO 2008/136838 and WO 2009/023232 teachthe use of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), andderivatives thereof such as 2-cyano-3,12-dioxoolean-1,9(11)-dien-28-oicacid methyl ester (CDDO-Me) and amide derivatives, for the treatment ofdiseases such as cancer, Alzheimer's disease, Parkinson's disease,inflammatory bowel diseases, and multiple sclerosis. Similarly, U.S.Pat. No. 6,974,801 and WO 2004/064723 teach the use of2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile (CNDDO),1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl) imidazole (CDDO-Im),1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-2-methylimidazole, and1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-4-methylimidazole inthe prevention or treatment of cancer, Alzheimer's disease, Parkinson'sdisease, multiple sclerosis, rheumatoid arthritis, and otherinflammatory diseases. Furthermore, the use of triterpenoids such asCDDO, CDDO-Me, CDDO-Im, and CDDO-Ethylamide in stimulating the growthand repair of bone and cartilage (US 2008/0233195 and WO 2008/064132) aswell as in inhibiting HIV-1 replication (WO 2005/046732) has beendescribed. US 2009/0326063 further teaches the use of synthetictriterpenoids in the prevention and treatment of renal/kidney disease,insulin resistance/diabetes, fatty liver disease, and/or endothelialdysfunction/cardiovascular disease.

Combination therapies of CDDO or CDDO-Me and a chemotherapeutic agent,immunosuppressive agent, or proteasome inhibitor are described in U.S.Pat. No. 7,435,755, U.S. Pat. No. 7,795,305, US 2009/0018146, US2009/0048205, WO 2002/047611 and WO 2009/023845 for the treatment ofcancer and graft versus host disease. Moreover, formulations forimproved oral bioavailability of CDDO-Me are disclosed in WO2010/093944.

Given the activity of CDDO and CDDO-Me, additional oleanolic acidderivatives have been developed for use in treating cancer,cardiovascular disease, neurodegenerative disease, renal/kidney disease,diabetes, arthritis and inflammatory conditions such as obesity,hypertension, atherosclerosis, coronary heart disease, stroke,peripheral vascular disease, hypertension, nephropathy, neuropathy,myonecrosis, ulcerative colitis, Crohn's disease, irritable bowelsyndrome, retinopathy and metabolic syndrome. See U.S. Pat. No.7,915,402, U.S. Pat. No. 7,943,778, US 2010/0048887, US 2010/0048892, US2010/0048911, US 2011/0245206 and US 2011/0245233.

SUMMARY OF THE INVENTION

The present invention is a triterpenoid compound of Formula I, or ahydrate, isomer, prodrug or pharmaceutically acceptable salt thereof:

wherein one or more of R¹, R² or R³ is independently a substituted orunsubstituted heteroaryl group, cycloalkyl group, heterocyclyl group,carboxamide group, nitrile group, haloalkyl group, or acyl group, andthe remaining R groups are hydrogen. In a particular embodiment, thecompound is provided in a pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amount of compounds of the invention, which remainedafter a 6 hour incubation in human plasma at 37° C.

FIG. 2A and FIG. 2B show the induction of cytoprotective enzymeexpression in mouse liver, kidney and lung. CB57BL/6 mice were gavagedwith 1 μmole of each triterpenoid. Six hours later, organs wereharvested, RNA was extracted, and mRNA levels for HO-1 (FIG. 2A) andNQO1 (FIG. 2B) were quantified by real-time PCR analysis. Results arepresented as the fold induction for each compound compared to thevehicle (DMSO) control. Data are presented as mean±SE of six mice pergroup.

DETAILED DESCRIPTION OF THE INVENTION

Pyridyl analogs of CDDO-Im 1 have now been developed, which are morestable in human plasma and achieve a higher concentration in targettissues such as liver, pancreas, kidney and lungs.

The compounds described herein are therefore of use in the treatment ofdisease, especially inflammatory diseases and cancer. Compoundsparticularly embraced by this invention have the structure of Formula I,which includes hydrates, isomers, prodrugs or pharmaceuticallyacceptable salts of Formula I:

wherein one or more of R¹, R² or R³ is independently a heteroaryl group,cycloalkyl group, heterocyclyl group, carboxamide group, nitrile group,haloalkyl group, or acyl group, each of which may be substituted orunsubstituted where appropriate, and the remaining R groups arehydrogen. In a particular embodiment, R² is a substituted orunsubstituted aryl group, heteroaryl group, cycloalkyl group orheterocyclyl group, and R¹ and R³ are hydrogen.

The term “heteroaryl” refers to a five- or six-membered aromatic ringstructure, wherein at least one of the aromatic ring atoms is nitrogen,oxygen or sulfur, and wherein the monovalent group is composed ofcarbon, hydrogen, aromatic nitrogen, aromatic oxygen or aromatic sulfur.Non-limiting examples of aryl groups include acridinyl, furanyl,imidazoimidazolyl, imidazopyrazolyl, imidazopyridinyl,imidazopyrimidinyl, indolyl, indazolinyl, methylpyridyl, oxazolyl,phenylimidazolyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl,quinazolyl, quinoxalinyl, tetrahydroquinolinyl, thienyl, triazinyl,pyrrolopyridinyl, pyrrolopyrimidinyl, pyrrolopyrazinyl,pyrrolotriazinyl, pyrroloimidazolyl, and chromenyl, wherein the point ofattachment is one of the aromatic atoms. In particular embodiments, theheteroaryl is a pyridyl group.

“Cycloalkyl” means a non-aromatic mono- or multicyclic ring systemincluding about 3 to about 10 carbon atoms, preferably about 5 to about10 carbon atoms. Non-limiting examples of suitable monocycliccycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyland the like. Non-limiting examples of suitable multicyclic cycloalkylsinclude 1-decalinyl, norbornyl, adamantyl and the like.

“Heterocyclyl” or “heterocycloalkyl” means a non-aromatic saturatedmonocyclic or multicyclic ring system including about 3 to about 10 ringatoms, preferably about 5 to about 10 ring atoms, in which one or moreof the atoms in the ring system is an element other than carbon, forexample nitrogen, oxygen or sulfur, alone or in combination. Preferredheterocyclyls contain about 5 to about 6 ring atoms. The prefix aza, oxaor thia before the heterocyclyl root name means that at least anitrogen, oxygen or sulfur atom respectively is present as a ring atom.The nitrogen or sulfur atom of the heterocyclyl can be optionallyoxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.Non-limiting examples of suitable monocyclic heterocyclyl rings includepiperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl,thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl,lactam, lactone, and the like. Non-limiting examples of suitablebicyclic heterocyclyl rings include decahydro-isoquinoline,decahydro-[2,6]naphthyridine, and the like.

As used herein, a “carboxamide” or “carboxamide group” refers to a—C(═O)NH₂ group.

The term “nitrile” or “nitrile group” is intended to refer to a —C≡Ngroup.

As used herein, “alkyl” or “alkyl group” includes linear or branchedsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. C₁₋₆ alkyl, for example, includes C₁, C₂, C₃, C₄, C₅, andC₆ alkyl groups. Examples of alkyl include, but are not limited to,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,t-butyl, n-pentyl, sec-pentyl, 3-(2-methyl)butyl, 2-pentyl,2-methylbutyl, n-hexyl, and 2-methylpentyl. In particular embodiments,an alkyl of this invention is a C₁₋₆ alkyl, C₁₋₅ alkyl, C₁₋₄ alkyl,alkyl, or C₁₋₂ alkyl.

The term “haloalkyl group” refers to a linear or branched alkyl groupsubstituted by one or more halogen atoms, the same or different,optionally selected from fluorine, chlorine, bromine, and iodine.Examples of this group include fluoromethyl, difluoromethyl,trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,2,2,2-trichloroethyl, 2,2,3,3-tetrafluoropropyl,2,2,3,3,3-pentafluoropropyl.

“Acyl,” as used herein alone or as part of another group, refers to a—C(═O)R radical, where R is, e.g., an aryl, alkyl, alkenyl, alkynyl, orcycloalkyl group.

The term “aryl” refers to a monovalent group with an aromatic carbonatom as the point of attachment, said carbon atom forming part of afive- or six-membered aromatic ring structure wherein the ring atoms areall carbon, and wherein the monovalent group is composed of carbon andhydrogen. Non-limiting examples of aryl groups include phenyl,methylphenyl, (dimethyl)phenyl, ethylphenyl, propylphenyl, —C₆H₄CH(CH₃)₂, —C₆H₄CH (CH₂)₂, methylethylphenyl, vinylphenyl, naphthyl, andthe monovalent group derived from biphenyl. In particular embodiments,the aryl is a phenyl group.

As used herein, “alkenyl” or “alkenyl group” refers to an unsaturatedbranched, straight-chain or cyclic monovalent hydrocarbon radical havingat least one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The radicalmay be in either the cis or trans conformation about the double bond(s).Examples of alkenyl include, but are not limited to, ethenyl, propenyls,such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-lyl, but-2-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien1-yl, beta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en3-yl, cyclobuta-1,3-dien-1-yl.

As used herein, an “alkynyl” or “alkynyl group” refers to an unsaturatedbranched, straight-chain or cyclic monovalent hydrocarbon radical havingat least one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Examples ofalkynyl groups include, but are not limited to, ethynyl, propynyl,butynyls, propargyl, and the like.

Any of the groups described herein may be unsubstituted or optionallysubstituted. When modifying a particular group, “substituted” means thatthe group the term modifies may, but does not have to, be substituted.Substitutions include the replacement of an available hydrogen with analkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl,heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl,hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, alkoxyalkoxy, acyl,halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl,aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,cycloalkyl, or heterocyclyl.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

Exemplary compounds within the scope of this invention include, but arenot limited to:

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, and whichpossess the desired pharmacological activity. Such salts include acidaddition salts formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or with organic acids such as 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid,3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylicacid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and di-carboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

Compounds of the invention may also exist in prodrug form. Sinceprodrugs are known to enhance numerous desirable qualities ofpharmaceuticals, e.g., solubility, bioavailability, manufacturing, etc.,the compounds employed in some methods of the invention may, if desired,be delivered in prodrug form. Thus, the invention contemplates prodrugsof compounds of the present invention as well as methods of deliveringprodrugs. Prodrugs of the compounds employed in the invention may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compound. Accordingly, prodrugs include, forexample, compounds described herein in which a hydroxy, amino, orcarboxy group is bonded to any group that, when the prodrug isadministered to a patient, cleaves to form a hydroxy, amino, orcarboxylic acid, respectively. For example, a compound comprising ahydroxy group may be administered as an ester that is converted byhydrolysis in vivo to the hydroxy compound. Suitable esters that may beconverted in vivo into hydroxy compounds include acetates, citrates,lactates, phosphates, tartrates, malonates, oxalates, salicylates,propionates, succinates, fumarates, maleates,methylene-bis-β-hydroxynaphthoate, gentisates, isethionates,di-p-toluoyltartrates, methanesulfonates, ethanesulfonates,benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates,esters of amino acids, and the like. Similarly, a compound comprising anamine group may be administered as an amide that is converted byhydrolysis in vivo to the amine compound.

A compound of this invention may be administered in a pharmaceuticalcomposition by various routes including, but not limited to, oral,subcutaneous, intravenous, or intraperitoneal administration (e.g. byinjection). Depending on the route of administration, the activecompound may be coated in a material to protect the compound from theaction of acids and other natural conditions which may inactivate thecompound.

For example, to administer the therapeutic compound by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation. By way of illustration, the therapeutic compound may beadministered to a subject in an appropriate carrier, for example,liposomes, or a diluent. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan, et al. (1984) J. Neuroimmunol. 7:27).

The compound may also be administered parenterally, intraperitoneally,intraspinally, or intracerebrally. Dispersions can be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compound can be orally administered, for example, with an inertdiluent or an assimilable edible carrier. The therapeutic compound andother ingredients may also be enclosed in a hard or soft shell gelatincapsule, compressed into tablets, or incorporated directly into thesubject's diet. For oral therapeutic administration, the therapeuticcompound may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a subject.

One or more compounds of the invention are administered at atherapeutically effective dosage sufficient to treat a condition in asubject. A “therapeutically effective dosage” preferably reduces theamount of symptoms of the condition in the infected subject by at leastabout 20%, more preferably by at least about 40%, even more preferablyby at least about 60%, and still more preferably by at least about 80%relative to untreated subjects. For example, the efficacy of a compoundcan be evaluated in an animal model system that may be predictive ofefficacy in treating the disease in humans.

The triterpenoid compounds of the invention are of use in modulatingIFN-γ-induced NO production in macrophages, said composition having anIC₅₀ value of at least less than 0.6 μM, more preferably less than 0.020μM.

In one embodiment, the instant compounds are of use in a method ofmodulating excessive nitric oxide or prostaglandin formation in asubject by administering to a subject a pharmaceutically effectiveamount of one or more triterpenoid compounds, such that the nitric oxideor prostaglandin formation is modulated.

In a further embodiment, the compounds of the invention are of use in amethod of preventing or treating a disorder characterized byoverexpression of iNOS or COX-2 genes, wherein the method includesadministering to a subject a pharmaceutically effective amount of one ormore triterpenoid compounds, such that the disorder is prevented ortreated. In a preferred embodiment, the disorder includes cancer,diabetic nephropathy, neurodegenerative disease, rheumatoid arthritis,inflammatory bowel disease, and other diseases whose pathogenesis isbelieved to involve excessive production of either nitric oxide orprostaglandins. In a particular embodiment, the neurodegenerativedisease includes Parkinson's disease, Alzheimer's disease, multiplesclerosis, and amyotrophic lateral sclerosis. The cancer may include,e.g., a leukemic cancer or a solid cancer. A leukemic cancer is a cancerof a blood cell, a myeloid cell, a monocytic cell, a myelocytic cell, apromyelocytic cell, a myeloblastic cell, a lymphocytic cell, or alymphoblastic cell. A solid cancer is a cancer of a bladder cell, abreast cell, a lung cell, a colon cell, a prostate cell, a liver cell, apancreatic cell, a stomach cell, a testicular cell, a brain cell, anovarian cell, a skin cell, a brain cell, a bone cell, or a soft tissuecell.

Moreover, the invention provides methods for the treatment andprevention of graft versus host disease (GVHD) by providing atriterpenoid compound of the invention either alone or in conjunctionwith another agent, such as an immunosuppressive agent such as acorticosteroid or tacrolimus, or a chemotherapeutic agent for thetreatment of GVHD. In graft versus host disease the donor immune systemmounts a response against the host's organs or tissue. As CDDOcompounds, either alone or in conjunction with other agents, can induceapoptosis by inhibiting Bcl-2 and have activity in lymphoid tissue, theinstant compounds can also be used to provide therapy for graft versushost diseases.

The practice of the methods of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Genetics; MolecularCloning A Laboratory Manual, 2nd Ed. (Sambrook, et al., ed. (1989)) ColdSpring Harbor Laboratory Press; Short Protocols in Molecular Biology,3rd Ed. (Ausubel, et al., ed. (1995)) Wiley, N.Y.; DNA Cloning, VolumesI and II (Glover, ed. (1985)); Oligonucleotide Synthesis (Gait, ed.(1984)); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Hames &Higgins, eds. (1984)); Immunochemical Methods In Cell And MolecularBiology (Mayer & Walker, eds. (1987)) Academic Press, London; HandbookOf Experimental Immunology, Volumes I-IV (Weir & Blackwell, eds.(1986)).

The invention is described in greater detail by the followingnon-limiting examples.

Example 1: Synthesis of Pyridine Analogs of CDDO-Im

The triterpenoids of the invention can be generally produced fromnatural compounds such as oleanolic acid, ursolic acid, betulinic acid,or hederagenin, or derivatives thereof that include additional A and/orC ring modifications. Synthesis of the compounds can be achieved usingany conventional method of synthesizing similar triterpenoids such asCDDO or CDDO-Me. See, e.g., U.S. Pat. No. 6,326,507, U.S. Pat. No.6,552,075, U.S. Pat. No. 6,974,801, U.S. Pat. Nos. 7,288,568, 7,863,327,U.S. Pat. No. 7,915,402, U.S. Pat. No. 7,943,778, U.S. Pat. No.8,034,955, U.S. Pat. No. 8,071,632, U.S. Pat. No. 8,124,656, U.S. Pat.No. 8,124,799, U.S. Pat. No. 8,129,429 and WO 2009/146216.

The synthesis of CDDO imidazolides 2-5 was achieved by directcondensation of CDDO-Cl and the corresponding 4-substituted imidazolidepyridyls with a yield between 50-98% (Table 1). The imadozilides,3-(1H-imidazol-4-yl)pyridine was obtained from a commercial source,while 4-(1H-imidazol-4-yl)pyridine and 2-(1H-imidazol-4-yl)pyridine weresynthesized by known procedures (Horne, et al. (1994) Heterocycles39:139-53; US 2010/026796).

TABLE 1

Compound R Yield (%) CDDO-Im 1 H  78* CDDO-Phenyl-Im 2 C₆H₅ 98CDDO-3P-Im 3 3-pyridyl 80 CDDO-2P-Im 4 2-pyridyl 95 CDDO-4P-Im 54-pyridyl 50 *Liby, et al. (2006) Clin. Cancer Res. 12: 4288-4293.

CDDO-Phenyl-Im 2 presents in its structure the phenyl-tetheredimidazolide while the three other analogues incorporate a pyridyl groupin the imidazolide in which the nitrogen position is variable.CDDO-3P-Im 3 possesses the nitrogen in meta (3 position), CDDO-2P-Im 4in ortho (2 position) and CDDO-4P-Im 5 in para (4 position). Thestructures and the purity of the new triterpenoids were confirmed by NMRspectral analyses.

Example 2: Pyridine Analogs Induce Monocytic Differentiation

CDDO-Im 1 is a potent inducer of differentiation in U937 cells (Place,et al. (2003) Clin. Cancer Res. 9:2798-2806). Therefore, the activity ofthe CDDO-Im analogs was evaluated using the U937 leukemia cell line.CD11b (also known as Mac-1α or CR3 complement receptor) was used as asurface marker of monocytic differentiation, and its expression wasmeasured by flow cytometry after 4 days of treatment with CDDO-Phenyl-Im2, CDDO-3P-Im 3, CDDO-2P-Im 4, CDDO-4P-Im 5, CDDO-Im 1 (as positivecontrol) or vehicle (DMSO). This analysis indicated that 30 nMCDDO-3P-Im was as effective as 30 nM CDDO-Im to induce differentiationof U937 cells (approximately a 3-fold induction of CD11b expressioncompared to the DMSO-treated cells), whereas CDDO-Phenyl-Im, CDDO-2P-Im,and CDDO-4P-Im were less active at this concentration. However,increasing the concentration to 100 nM enhanced the activity ofCDDO-2P-Im and CDDO-4P-Im. Indeed, the highest level of differentiationwas obtained using 100 nM of CDDO-2P-Im (approximately a 4-foldinduction of CD11b).

As CDDO-Im and CDDO-3P-Im at 100 nM induced cell death in thedifferentiation assay, U937 cells were treated for 48 hours to measureapoptosis. Both CDDO-Im and CDDO-3P-Im increased annexin V staining, amarker of early apoptosis.

Example 3: Pyridine Analogs Inhibit NO Production

Because oxidative and inflammatory stress contribute to carcinogenesis(Albini & Sporn (2007) Nature Rev. Cancer 7:139-147), it was determinedwhether the pyridine analogues could block de novo synthesis ofinducible nitric oxide synthase (iNOS), a critical enzyme involved inthe inflammatory response (Kroncke, et al. (1998) Clin. Exp. Immunol.113:147-156; Zamora, et al. (2000) Mol. Med. 6:347-373). Nitric oxiderelease was determined in RA W264.7 macrophage-like cells, afterstimulation with 10 ng/ml interferon-γ (IFNγ) and a 24 hour-exposure toeach compound at 0.625, 1.3, 2.5, 5, 10 and 20 nM. NO release wasmeasured by Griess reaction and compared to CDDO-Im, which is a potentsuppressor of iNOS (Place, et al. (2003) Clin. Cancer Res.19:2798-2806). This analysis indicated that the new analogues wereslightly less potent than CDDO-Im for blocking NO production. However,each analog inhibited NO production in the low nanomolar range with IC₅₀values between 2-15 nM (Table 2). The order of potency in the NO assaywas similar to the results obtained in the U937 differentiation assaywith CDDO-Im (2.0 nM) and CDDO-3P-Im (4.3 nM) being the most active andCDDO-Phenyl-Im being the least active (14.7 nM).

TABLE 2 IC₅₀ (nM) Compound Mean ± SE CDDO-Im 1 2.0 ± 0.6 CDDO-Phenyl-Im2 14.7 ± 2.9  CDDO-3P-Im 3 4.3 ± 0.7 CDDO-2P-Im 4 5.8 ± 1.1 CDDO-4P-Im 59.0 ± 0.6

Example 4: Stability of Pyridine Analogs in Human Serum

Since CDDO-Im is not stable in human plasma, the stability of theCDDO-Im analogs was determined. Compounds were incubated in human plasmaat 37° C. for up to 6 hours. The compounds were extracted withacetonitrile at six different time points (0, 0.5, 1, 2, 4 and 6 hours),centrifuged, and the percent of starting material remaining in thesupernatant was measured by liquid chromatography-mass spectrometry(LC-MS). This analysis indicated that CDDO-Im was rapidly metabolized;more than 88% of the CDDO-Im was lost within 30 minutes, and this lossincreased to 98% within the next 5 hours (FIG. 1). In contrast, thedegradation graphs of the CDDO-Im analogs revealed greater stabilitycompared to CDDO-Im (FIG. 1). For the least stable compound, CDDO-4P-Im,42% of the starting material was still detected after a 1-hourincubation, while more than 85% of CDDO-2P-Im remained in plasma. Theconcentrations then decreased gradually over 6 hours. CDDO-2P-Im was themost stable analog with more than 50% left in plasma after 4 hours.CDDO-Phenyl-Im, CDDO-3P-Im, and CDDO-4P-Im were all more stable thanCDDO-Im. Further LC-MS analysis on the samples revealed that CDDO-Im wasessentially converted to its parent molecule2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid (CDDO), whoseconcentration increased over time in human plasma. Unexpectedly, theCDDO-Im analogs are metabolized differently from CCDO-Im, as no tracesof CDDO were detected with any of the other compounds at any time point.Instead, an oxidized derivative was detected on the chromatogram, in theform of an adduct on the molecule of +16 in MW (N-oxide).

Example 5: Tissue Distribution CDDO-Im Pyridine Analogs

To determine concentrations of the CDDO-Im analogs in various organs, anin vivo evaluation was conducted in C57BL/6 mice. Six hours after asingle gavage with 1 μmole of compound, the mice were sacrificed and thetissues were collected. Table 3 shows concentrations measured in theliver, pancreas, kidney, lung, whole blood and plasma of mice by LC-MS.

TABLE 3 Compound Concentration (μmole/kg) Mean ± SE Tissue 1 2 3 4 5Liver  1.7 ± 0.32 12.2 ± 1.27  5.9 ± 0.57 14.9 ± 2.58  2.8 ± 0.47Pancreas 0.31 ± 0.09 1.97 ± 0.19 2.52 ± 0.24 3.55 ± 0.39 0.30 ± 0.04Kidney 0.24 ± 0.08 5.79 ± 0.25 1.47 ± 0.24 3.70 ± 0.55 0.35 ± 0.06 Lungs0.19 ± 0.05 1.28 ± 0.17 2.49 ± 0.44 2.63 ± 0.41 0.17 ± 0.05 Whole Blood0.03 ± 0.01 0.35 ± 0.03 0.16 ± 0.02 0.53 ± 0.02 0.04 ± 0.1  Plasma 0.06± 0.02 0.12 ± 0.01 0.15 ± 0.04 0.52 ± 0.02 0.03 ± 0.01

The bioavailability of CDDO-3P-Im 3, CDDO-2P-Im 4 and CDDO-Phenyl-Im 2was better than for CDDO-Im 1 as demonstrated by the higher drugconcentrations attained in each organ. In contrast, levels obtained withCDDO-4P-Im 5 were similar to CDDO-Im 1. The highest levels for all ofthese compounds were detected in the liver with concentrations of 12-15μM for CDDO-2P-Im 4 and CDDO-Phenyl-Im 2 vs. 1.7 μM for CDDO-Im 1. Inkidneys, the highest concentration was obtained with CDDO-Phenyl-Im 2(5.8 μM), followed by CDDO-2P-Im 4, CDDO-3P-Im 3, and CDDO-4P-Im 5. Verylow concentrations of CDDO-Im 1 (approximately 0.3 μM) were detected inthe pancreas, with much higher levels obtained with CDDO-2P-Im 4 (3.5μM), CDDO-3P-Im 3 (2.5 μM), and CDDO-Phenyl-Im 2 (2 μM). Lower tissuelevels were found in lungs for all of the compounds compared to theliver and kidney, but drug levels in the lungs were greater than 2.5 μMfor CDDO-3P-Im 3 and CDDO-2P-Im 4 vs. 0.2 μM for the parental CDDO-Im 1.The concentration of triterpenoids was higher in whole blood than inplasma. While the detection of transformation products was also carriedout in plasma, no traces of any oxidized forms could be found,suggesting a distinct metabolization in mice vs. humans.

Example 6: CDDO-Im Pyridine Analogs Induce HO-1 and NQO1 Expression InVivo

Synthetic triterpenoids such as CDDO-Im strongly activate the Nrf2/AREpathway; Nrf2 is a transcription factor that regulates numerouscytoprotective and anti-inflammatory genes (Liby, et al. (2005) CancerRes. 65:4789-98; Yates, et al. (2007) Mol. Cancer Therapeut. 6:154-62).Therefore, the ability of the new pyridine compounds to inducetranscription of the Nrf2 target genes HO-1 and NQ01 in vivo wasassessed. In tissues harvested from mice six hours after gavage with thedrugs, an increase in the expression of HO-1 was observed with alltriterpenoids, compared to the vehicle control (FIG. 2A). In the liver,CDDO-3P-Im and CDDO-2P-Im induced high levels of HO-1, which were about3 times more elevated than with CDDO-Im (13.4- and 15.2-fold,respectively vs. 4.9-fold for CDDO-Im). CDDO-Phenyl-Im and CDDO-4P-Impresented similar induction of HO-1 compared to the parent molecule.While induction levels were lower in the kidney than in the liver, theoverall profile in the kidney was similar, except that CDDO-Phenyl-Imwas markedly less potent. The results obtained in lungs were, on theother hand, unexpected. Although CDDO-4P-Im did not accumulate in lungsbased on the pharmacokinetic study summarized in Table 3, it notablyinduced gene expression in the lungs. HO-1 transcript levels wereincreased almost 3 times more than with CDDO-Im or any of the othercompounds, while it was less potent than CDDO-Im in the liver andkidney.

Because HO-1 is regulated by transcription factors in addition to Nrf2(Alam, et al. (1999) J. Biol. Chem. 274:26071-8; Kensler, et al. (2007)Ann. Rev. Pharmacol. Toxicol. 47:89-116), induction of the prototypicalNrf2 target gene NQO1 was also examined. All of the CDDO-Im analogsinduced the expression of NQO1 mRNA (FIG. 2B), although they wereslightly less potent than CDDO-Im in the liver, with the exception ofCDDO-Phenyl-Im which was markedly less potent than CDDO-Im. The sametrend of NQO1 mRNA induction was observed both in liver and kidney, withCDDO-Im as the most potent compound, followed by CDDO-2P-Im, CDDO-3P-Im,CDDO-4P-Im and then CDDO-Phenyl-Im. In lungs, the pyridine analogs wereless active than CDDO-Im but all displayed a 2.4- to 4-fold increase ofNQO1 mRNA.

To confirm those findings, immunoblot analysis was performed for HO-1.Protein extracts were prepared from the homogenized mouse livers,kidneys and lung, and subsequently examined by western blot. Thisanalysis indicated that HO-1 protein levels were increased in parallelwith mRNA transcript levels.

What is claimed is:
 1. A triterpenoid compound having the structure ofFormula I, or a hydrate, stereoisomer, prodrug or pharmaceuticallyacceptable salt thereof:

wherein one or more of R¹, R² or R³ is independently a substituted orunsubstituted carboxamide group and the remaining R groups are hydrogen.2. A pharmaceutical composition comprising the compound of claim 1 or ahydrate, stereoisomer, or pharmaceutically acceptable salt thereof inadmixture with a pharmaceutically acceptable carrier.
 3. Thetriterpenoid compound of claim 1 or a hydrate, stereoisomer, orpharmaceutically acceptable salt thereof, wherein R¹ is a substituted orunsubstituted carboxamide and R² and R³ are hydrogen.
 4. Thetriterpenoid compound of claim 1 or a hydrate, stereoisomer, orpharmaceutically acceptable salt thereof, wherein R² is a substituted orunsubstituted carboxamide and R¹ and R³ are hydrogen.
 5. Thetriterpenoid compound of claim 1 or a hydrate, stereoisomer, orpharmaceutically acceptable salt thereof, wherein R³ is a substituted orunsubstituted carboxamide and R¹ and R² are hydrogen.
 6. Thetriterpenoid compound of claim 1 or a hydrate, stereoisomer, orpharmaceutically acceptable salt thereof, wherein the compound is


7. A method of treating disorder characterized by overexpression of iNOSor COX-2 genes, the method comprising administering to a subject in needthereof a pharmaceutically effective amount of the triterpenoid compoundof claim 1 or a hydrate, stereoisomer, or pharmaceutically acceptablesalt thereof.
 8. The method of claim 7, wherein the disorder is cancer.9. A triterpenoid compound having the structure of Formula I, or ahydrate, stereoisomer, prodrug or pharmaceutically acceptable saltthereof:

wherein one or more of R¹ or R³ is independently a substituted orunsubstituted nitrile group, haloalkyl group, or acyl group, and theremaining R groups are hydrogen.
 10. The triterpenoid compound of claim9 or a hydrate, stereoisomer, or pharmaceutically acceptable saltthereof, wherein R¹ is a substituted or unsubstituted nitrile and R² andR³ are hydrogen.
 11. The triterpenoid compound of claim 9 or a hydrate,stereoisomer, or pharmaceutically acceptable salt thereof, wherein R³ isa substituted or unsubstituted nitrile and R¹ and R² are hydrogen. 12.The triterpenoid compound of claim 9 or a hydrate, stereoisomer, orpharmaceutically acceptable salt thereof, wherein R¹ is a substituted orunsubstituted haloalkyl and R² and R³ are hydrogen.
 13. The triterpenoidcompound of claim 9 or a hydrate, stereoisomer, or pharmaceuticallyacceptable salt thereof, wherein R³ is a substituted or unsubstitutedhaloalkyl and R¹ and R² are hydrogen.
 14. The triterpenoid compound ofclaim 9 or a hydrate, stereoisomer, or pharmaceutically acceptable saltthereof, wherein R¹ is a substituted or unsubstituted acyl and R² and R³are hydrogen.
 15. The triterpenoid compound of claim 9 or a hydrate,stereoisomer, or pharmaceutically acceptable salt thereof, wherein R³ isa substituted or unsubstituted acyl and R¹ and R² are hydrogen.
 16. Apharmaceutical composition comprising the compound of claim 9 or ahydrate, stereoisomer, or pharmaceutically acceptable salt thereof inadmixture with a pharmaceutically acceptable carrier.
 17. A method oftreating disorder characterized by overexpression of iNOS or COX-2genes, the method comprising administering to a subject in need thereofa pharmaceutically effective amount of the triterpenoid compound ofclaim 9 or a hydrate, stereoisomer, or pharmaceutically acceptable saltthereof.
 18. The method of claim 17, wherein the disorder is cancer. 19.A triterpenoid compound or a hydrate, stereoisomer, or pharmaceuticallyacceptable salt thereof, wherein the compound is selected from the groupconsisting of: